The importance of developing a better understanding of the function and regulation of cytochrome c oxidase has become increasingly apparent, given its decisive influence on mitochondrial metabolism and the central role of mitochondria in controlling cell life and death. Research supported by this grant has led us to a new perspective on this complex energy conserving machine, derived from a number of new high resolution structures of the enzyme from the mitochondrial model system, Rhodobacter sphaeroides. These reveal previously unobserved changes in conformation associated with altered redox state, and the presence of lipid and steroid binding sites conserved in bacteria and mammals. This proposal is aimed at determining the significance of the novel structural findings through further crystallographic efforts designed to obtain new and higher resolution crystal forms, and through studies of the effects of lipidic ligands on activity, stability and efficiency of oxidase. The Specific Aims are: 1) to generate additional crystal forms of two and four subunit Rhodobacter oxidase, using molecular engineering strategies and robotic crystal screening; 2) to create, characterize and crystallize mutants that facilitate the trapping of novel catalytic intermediates or that restrain flexibility, to look for new conformational states and test the importance of conformational change; 3) to screen for alternative ligands of a steroid binding site, with potential physiological significance, or inhibitory or stabilizing effects. A major tool in these studies will be crystallography, but our ability to comprehensively analyze oxidase function and spectral features, including on-line crystal spectra, will be crucial to interpreting the structural findings. The expected outcome is a new level of understanding of the molecular mechanism of energy conversion in cytochrome oxidase, including the role of conformational change in gating and efficiency, and the regulatory effects of lipidic ligands. The long term goal is to better understand the involvement of cytochrome oxidase in metabolic disease states including cancer, obesity, diabetes and aging, through structure/function analysis and the discovery of compounds that are physiological effectors, crystallization aids, mechanistic probes, or precursors to drugs that can modulate oxidase activity.